Prostate cancer is the second leading cause of cancer death in American men. To date, there is no cure for the disease once it has reached a metastatic, castration-resistant state. Therapies that increase survival by several months are considered clinical successes. Alternative approaches to treatment are obviously necessary. The studies outlined in this proposal will address this need by exploiting a novel and underexplored drug delivery vehicle, the pH(Low) Insertion Peptide (pHLIP), for tumor selective delivery of clinically validated cytotoxic monomethyl auristatins (MMAs) in the form of pHLIP-MMA conjugate. Unlike antibody drug conjugates, pHLIP's targeting and payload delivery mechanism relies solely on tumor acidity rather than on any specific antigen. This unique mechanism overcomes the need to escape endosomes or lysosomes and circumvents loss of antigen expression; drawbacks of antibody drug conjugates. Thus, we hypothesize that pHLIP-based drug conjugates will have an enhanced therapeutic window as compared to free drug, providing a mechanism to overcome the hurdle of systemic toxicity and ultimately improve patient outcome over current chemotherapy regimens. A series of experiments have been outlined to specifically address the diverse efforts necessary for each of the steps in the pHLIP-MMA development process. Aim 1 will concentrate on biophysical and biochemical characterization of pHLIP-MMA conjugates to optimize in vitro cytotoxicity efficacy in prostate cancer cell lines. Efforts will focus on cytotoxic agent, linker nd peptide design, and stability as variables for enhancing efficacy. Aim 2 will test the therapeutic efficacy associated with the lead pHLIP-MMA conjugate identified in Aim 1 through biodistribution, pharmacokinetics, maximum tolerated dose, and magnetic resonance imaging of tumor volume studies in orthotopic and bone metastasis models of castration-resistant prostate cancer. Successful completion of the proposed work will result in a lead molecule with sufficient data to warrant additional development along a path to clinical testing.